Acute lung injury (ALI) is characterized by increased pulmonary endothelial permeability and pulmonary edema triggered by reactive oxygen species (ROS) and injurious cytokines. Endothelial dysfunction includes increased paracellular permeability to water and protein as well as an increase in endothelial cell death by both necrosis and apoptosis. Apoptosis is a form of programmed cell death with distinct biochemical and morphological features. Increasing evidence has implicated endothelial apoptosis as a major component of acute lung injury but the regulation of pulmonary endothelial apoptosis remains poorly understood. Studies in some systemic endothelial cells have identified the signaling molecule cyclic guanosine monophosphate (cGMP) as a pro-survival, anti-apoptotic factor whereas in other cells cGMP triggers apoptosis. cGMP is generated by endothelial soluble guanylyl cyclase (sGC) after stimulation with nitric oxide (NO). cGMP modulates pulmonary endothelial permeability through activation of protein kinase G (PKG) but any effects on pulmonary endothelial apoptosis are unknown. This information is important because lung NO is increased by cyclic ventilatory stretch and the inflammation in ALI and inhaled NO is administered to improve oxygenation. The overall goal of this grant application is to determine the effect of cGMP on pulmonary endothelial apoptosis. Our preliminary data suggest that increases in human and mouse pulmonary endothelial cGMP concentration significantly attenuate ROS-triggered apoptosis. In Aim 1, the effect of modulating endothelial cGMP concentration on ROS-induced cell death will be examined in human and mouse lung microvascular endothelial cells. cGMP will be increased in endothelial monolayers by administration of membrane-permeant analogues, direct stimulation of sGC with Bay 41-2772, and physiologic sGC stimulation from 5% cyclic stretch; depletion of cGMP will be accomplished with sGC inhibition. In Aim 2, the molecular mechanisms of the anti-apoptotic effects of cGMP will be explored. The role of PKG will be determined using human pulmonary artery endothelial cells and pulmonary microvascular cells from wild-type and PKG knock-out mice. Downstream mechanisms will be explored, including possible upregulation of the pro-survival genes thioredoxin (Trx) and heme oxygenase 1 (HO-1). Acute lung injury is a common cause of lung failure in critically ill patients. Part of the tissue injury in this process includes a process of programmed cell death that may contribute to lung dysfunction. An understanding of how this phenomenon is regulated in acute lung injury may lead to new therapies for this severe illness. [unreadable] [unreadable] [unreadable] [unreadable]